Prostate cancer now ranks as the most prevalent cancer in men. Approximately 160,000 new cases are diagnosed each year; of these new cases, 35,000 will die of metastatic disease. In women, breast cancer kills 45,000 women per year. Steroid hormone receptors and the factors that bind steroid hormone receptors are key players in the maintenance of healthy tissue. Similarly, disregulation of steroid hormone receptors and steroid hormone receptor interacting proteins are important to the development of a wide variety of sex steroid hormone dependent cancers and diseases. Current therapies for such afflictions include surgery (e.g., castration) and chemical treatment (e.g., chemotherapy and hormone ablation therapy).
Androgens in normal prostate epithelium appear to primarily drive differentiation. In contrast, prostate cancer growth is directly androgen stimulated. Thus, one common therapy for the treatment of prostate cancer is androgen ablation therapy, to which most patients respond. Unfortunately, virtually all prostate cancer relapse is clinically androgen independent. Significantly, most androgen independent tumors express high levels of androgen receptor as well as androgen receptor regulated genes, indicating that the androgen receptor is transcriptionally active (van der Kwastet et al., Int. J. Cancer 48, 189-193 (1991); Ruizeveld de Winter et al., Am. J. Pathol. 144, 735-746 (1994); Taplin et al., N. Engl. J. Med. 332, 1393-1398 (1995); Hobisch et al., Cancer Res. 55, 3068-3072 (1995); Visakorpi et al., Nat. Genet. 9, 401-406 (1995); and Koivisto et al., Cancer Res. 57, 314-319 (1997)).
It has been demonstrated that structural changes in the androgen receptor contribute to altered androgen receptor function in primary or androgen independent prostate cancer. Most reports find that the androgen receptor is wild-type in primary androgen dependent prostate cancer, with a few exceptions (Tilley et al., Clin. Cancer Res. 2, 277-285 (1996)). In contrast, androgen receptor mutations have been identified in androgen independent prostate cancer (Taplin et al., supra; Culig et al., Mol. Endocrinol. 7, 1541-1550 (1993); Suzuki et al., J. Steroid Biochem. Mol. Biol. 46, 759-765 (1993); Suzuki et al., Prostate 29, 153-158 (1996); and Taplin et al., Cancer Res. 59, 2511-2515. (1999)). An analysis of androgen independent prostate cancer from a large number of bone marrow metastases recently showed that androgen receptor ligand binding domain mutations occur primarily in patients treated with the androgen receptor antagonist flutamide (Taplin et al., supra). Importantly, these mutations result in androgen receptors that are strongly stimulated by hydroxyflutamide. Fortunately, patients with these mutations respond to subsequent treatment with bicalutamide, an androgen receptor antagonist that remains active against these mutant androgen receptors (Taplin et al., supra; and Joyce et al., J. Urol. 159, 149-153 (1998)).
The above results indicate that additional mechanisms must contribute to androgen independent androgen receptor activity in the majority of patients treated with androgen ablation. For example, androgen receptor function is modulated by a growing list of associated proteins, some of which likely contribute to prostate cancer development or progression. Some of these proteins function as transcriptional co-activators through intrinsic histone acetyltransferase activity, association with CBP/p300, and/or binding to components of the RNA polymerase II complex (Onate et al., Science 270, 1354-1357 (1995); Hong et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4948-4952 (1996); Voegel et al., EMBO J. 15, 3667-3675 (1996); Anzick et al., Science 277, 965-968 (1997); Torchia et al., Curr. Opin. Cell Biol. 10, 373-383 (1998); Kamei et al., Cell 85, 403-414 (1996); and Spencer et al., Nature 389, 194-198 (1997)), but their roles and the roles of other androgen receptor associated proteins in prostate cancer remain unclear.
There remains a need for additional therapies for steroid hormone related carcinomas and other steroid hormone-related diseases. A better understanding of androgen receptor-associated cellular communication could greatly facilitate the discovery of drugs and therapeutic methods for the treatment of a broad range of conditions with fewer of the serious and variable side effects prevalent with currently available chemotherapeutic reagents and surgical procedures. Novel agonists and antagonists of the androgen receptor pathway would be invaluable to the field of therapeutics for steroid hormone receptor-related ailments.
The present invention provides a substantially pure nucleic acid molecule encoding a steroid hormone-interacting p21-activated kinase (PAKSI) polypeptide. Preferably, the nucleic acid molecule has the nucleic acid sequence of SEQ ID NO: 1. The invention also provides a substantially pure PAKSI polypeptide. Preferably, the substantially pure PAKSI polypeptide has the amino acid sequence of SEQ ID NO: 2. In a related aspect, the present invention provides a therapeutic composition that includes a PAKSI polypeptide formulated in a physiologically acceptable carrier.
The present invention also provides a method of inhibiting a steroid hormone receptor by administering to an individual a compound that is capable of inhibiting PAKSI. The compound may be capable of inhibiting PAKSI expression or PAKSI activity. In a related aspect, the method further provides a method of inhibiting a prostate tumor or a breast tumor by contacting the tumor with a compound that is capable of inhibiting PAKSI.
Alternatively, the present invention provides a method of stimulating the beneficial effects of a steroid hormone receptor by administering to an individual a PAKSI agonist. PAKSI agonists can also be used to treat and prevent other steroid hormone-related diseases. In one preferred embodiment, a PAKSI agonist can be administered to an individual to stimulate the therapeutic effects of an estrogen. For example, a PAKSI agonist can be administered to a patient diagnosed with cardiovascular disease to promote vasodilation. Thus, the present invention provides a method of inhibiting cardiovascular disease by administering to an individual a PAKSI agonist.
A particularly valuable aspect of the invention is that it provides for the identification of PAKSI modulatory compounds that may serve as useful therapeutics. Accordingly, the present invention provides a method of determining whether a compound is a PAKSI modulatory compound. The method involves the steps of: (a) providing a cell expressing a PAKSI polypeptide; (b) contacting the cell with a compound; and (c) measuring the expression or enzymatic-activity of PAKSI by the cell. An alteration in the level of the expression or activity indicates that the compound is a PAKSI modulatory compound.
Alternatively, a PAKSI modulatory compound can be identified by the steps of: (a) providing a PAKSI polypeptide or a PAKSI polypeptide fragment having PAKSI enzymatic activity; (b) contacting the PAKSI polypeptide or a PAKSI polypeptide fragment with the compound; and (c) measuring the enzymatic-activity of PAKSI. An alteration in the level of the activity of PAKSI indicates that the compound is a PAKSI modulatory compound. The PAKSI polypeptide or a PAKSI polypeptide fragment can be a recombinant polypeptide or polypeptide fragment. This method for the identification of a PAKSI modulatory compound can be performed in vitro.
The present invention further provides a method of diagnosing a mammal, preferably a human, for the presence of prostate cancer. Alternatively, the present invention provides a method of determining whether a mammal has an increased likelihood of developing prostate cancer. The method involves measuring PAKSI gene expression in a sample (e.g., of prostate tissue) from a mammal to determine whether an alteration in PAKSI expression has occurred relative to the PAKSI expression in a sample from an unaffected mammal. An alteration in PAKSI gene expression would be an indication that the mammal has prostate cancer, or an increased likelihood of developing prostate cancer. Alternatively, an alteration in PAKSI gene expression can be measured in a breast tissue sample and compared to a sample taken from an unaffected mammal, to diagnose the presence of breast cancer, or the likelihood of developing breast cancer.
Alternatively, the present invention provides a method of diagnosing a mammal for the presence of prostate cancer, or an increased likelihood of developing prostate cancer, by measuring PAKSI polypeptide enzymatic activity. The method involves (a) collecting a sample from a mammal; (b) measuring the PAKSI enzymatic activity of the sample; and (c) comparing the measured PAKSI enzymatic activity with the relative activity in a sample from an unaffected individual. An alteration in PAKSI enzymatic activity relative to a sample from an unaffected mammal is an indication that the mammal has prostate cancer or an increased likelihood of developing prostate cancer. Of course, this method is also applicable to diagnosis of other steroid hormone receptor-related diseases (e.g., diagnosis of breast cancer).
The present invention also provides a kit for diagnosing a mammal for the presence of a steroid hormone receptor-related disease. The kit may contain, for example, a panel of probes and/or primers specific to the PAKSI gene that can be used to measure the level of PAKSI mRNA expression in a mammal compared to PAKSI expression in a unaffected mammal. Alternatively, the kit may contain assay reagents useful for determining the level of PAKSI enzymatic activity in a sample from a mammal compared to the enzymatic activity of PAKSI in an unaffected mammal.
Finally, the present invention provides methods of treating a patient with a disease characterized by abnormal cell growth or by an abnormal cytoskeleton in a specific cell type by administering a PAKSI modulatory compound to the patient. In one example, the disease characterized by abnormal cell growth is cancer.
By xe2x80x9cPAKSI genexe2x80x9d is meant a gene encoding a polypeptide having PAKSI steroid hormone receptor binding activity and/or kinase activity. A PAKSI gene is a gene encoding a PAKSI polypeptide having about 60% or greater, or more preferably 70% or greater amino acid sequence identity to the PAKSI polypeptide disclosed herein (SEQ ID NO: 2). For example, the gene may encode human or murine PAKSI polypeptide. A PAKSI gene may also be defined as encoding a polypeptide with at least 50% of the activity of the PAKSI polypeptides described below.
xe2x80x9cPolypeptidexe2x80x9d means any chain of amino acids regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
xe2x80x9cSubstantially identicalxe2x80x9d means a polypeptide or nucleic acid exhibiting at least 60%, preferably 70%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 or 35 amino acids, and most preferably the full length polypeptide sequence. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 or 110 nucleotides, and most preferably the full length nucleic acid sequence.
Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
xe2x80x9cTransformed cellxe2x80x9d means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) a polypeptide described herein (for example, a PAKSI polypeptide).
xe2x80x9cTransformationxe2x80x9d means any method for introducing foreign molecules into a cell. For example, molecules may be introduced using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., chloroplasts and mitochondria), bacteria, yeast, fungi, algae, and animal tissue. Alternatively, standard methods of transformation, such as, calcium phosphate precipitation, DEAE dextran, lipofection, and virus-mediated transduction, can be used to transform a cell.
xe2x80x9cPurified antibodyxe2x80x9d means an antibody that is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., a PAKSI-specific antibody. A purified PAKSI antibody may be obtained, for example, by affinity chromatography using recombinantly-produced PAKSI protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a protein that is a PAKSI-related protein, but that does not substantially recognize and bind other molecules in a sample, e.g., a biological sample that naturally includes other proteins.
By xe2x80x9cmutantxe2x80x9d is meant different from what normally appears, occurs, or functions. As used herein, the term refers to a nucleic acid sequence which differs from the wild-type nucleic acid sequence. This term also describes a protein encoded by the mutant nucleic acid sequence. The term also means an organism which carries a mutant nucleic acid sequence.
By xe2x80x9cbiological activityxe2x80x9d or xe2x80x9cenzymatic activityxe2x80x9d0 is meant functional events mediated by a protein. In some embodiments, this includes assaying the amount of PAKSI binding to a steroid hormone receptor, or events assayed by measuring the biochemical events downstream of PAKSI binding a steroid hormone receptor (e.g., an androgen receptor or an estrogen receptor). It also includes interactions of a polypeptide with another polypeptide. The biological activity of PAKSI also includes modulations in cell function, for example, cell growth, cell survival, cell motility, and the structure and function of the cytoskeleton.
By xe2x80x9cactivating a steroid hormone receptorxe2x80x9d is meant, increasing the activity of a steroid hormone receptor above the level of activity in a wild-type cell.
By xe2x80x9cactivating the therapeutic effects of estrogenxe2x80x9d is meant, stimulating the biochemical effect of estrogen (e.g., by stimulating the estrogen receptor itself or stimulating an estrogen-receptor-related biochemical cascade) in a cell by contacting the cell with a compound, e.g., a PAKSI agonist.
By xe2x80x9csteroid hormone receptorxe2x80x9d is meant an androgen receptor, estrogen receptors (alpha and beta), progesterone receptor, glucocorticoid receptor, and other members of the nuclear receptor family that are regulated by steroid hormone binding. PAKSI may also interact with other nuclear receptors, such as the thyroid hormone receptor or retinoic acid receptors, which bind to ligands other than steroid hormones.
By xe2x80x9ccardiovascular diseasexe2x80x9d is meant, vasoconstriction, atherosclerosis, abnormal angiogenesis, thrombosis, stroke, myocardial infarction, pulmonary embolism, deep-vein thrombosis, transplant-associated vasculopathy, stenosis (e.g., vein graft stenosis or peri-anastomatic prosthetic graft stenosis), restenosis (e.g., restenosis after angioplasty or stent placement, and the like), atheroma, and vasculitis. Cardiovascular disease also refers to vascular conditions that develop after a surgical treatments, such as venous bypass surgery, balloon angioplasty, post-angioplasty of atherosclerotic plaques of both coronary and peripheral arteries, and allo- and xenograft rejection. Alternatively, cardiovascular disease is used to refer to the disease of a patient that has suffered ischemia, reperfusion injury, mechanical injury, immunologic injury, pharmacologic injury of a vessel, or coronary trauma.
By xe2x80x9cunaffected mammalxe2x80x9d is meant a mammal that does not have a steroid hormone receptor-related disease. For example, the mammal may lack any symptoms of prostate cancer or breast cancer, or would not otherwise benefit from steroid hormone related treatments.
By xe2x80x9cmodulate,xe2x80x9d xe2x80x9cmodulatory,xe2x80x9d or xe2x80x9cmodified,xe2x80x9d as, used herein, is meant increasing or decreasing the biological activity of PAKSI. Preferably the biological activity is increased or decreased 50% relative to a control. More preferably, the biological activity is increased or decreased 90% relative to a control. Most preferably, the biological activity is increased or decreased 95% relative to a control.
By xe2x80x9cassayingxe2x80x9d is meant analyzing the effect of a treatment or exposure, be it chemical or physical, administered to whole animals or cells derived therefrom. The material being analyzed may be an animal, a cell, a tissue, a lysate or extract derived from a cell, or a molecule derived from a cell. The analysis may be, for example, for the purpose of detecting, altered gene expression, altered nucleic acid stability (e.g., mRNA stability), altered protein stability, altered protein levels, or altered protein biological activity. The means for analyzing may include, for example, assaying for PAKSI message or protein, PAKSI kinase activity, or PAKSI steroid hormone receptor binding (i.e., by methods described herein). Such methods include gene analysis to detect alteration (mutation, loss or amplification) or polymorphisms of the PAKSI gene, RNA hybridization (Northern blot or in situ hybridization) for PAKSI message, PCR amplification for PAKSI message, immunological detection of PAKSI using specific antibodies (by immunoblotting, enzyme linked immunoassay, or immunohistochemistry), functional assays including kinase activity, association with other proteins, cell growth, cell survival, cell motility, alterations in the cytoskeleton of a cell, or PAKSI mediated phosphorylation of other proteins.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription of an operably-linked gene. Also included in the invention are those promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific or that are inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
xe2x80x9cOperably linkedxe2x80x9d means that a gene and a regulatory sequence (or sequences) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cconstitutively active,xe2x80x9d as referred to herein, is meant a nucleic acid sequence that encodes a polypeptide, which when expressed is in an active form at least as often, or more often as the wild-type polypeptide, in a cell in which wild-type polypeptide is naturally expressed. The polypeptide may be in an active form by being phosphorylated, dephosphorylated, cleaved from a propeptide to a peptide, or through a ligand independent mutation.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell or organism that includes a DNA sequence that is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organism is generally a transgenic non-human mammalian (e.g., rodents such as rats or mice) or invertebrate (e.g., Caenorhabditis elegans), and the DNA (transgene) is inserted by artifice into the genome.
xe2x80x9cTransgenexe2x80x9d means any piece of DNA that is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from that cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
xe2x80x9cConserved regionxe2x80x9d means any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity between two or more of the PAKSI family members.
xe2x80x9cDetectably-labeledxe2x80x9d means any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P or 35S) and nonradioactive labeling (e.g., chemiluminescent labeling, e.g., fluorescein labeling).
By xe2x80x9cantisensexe2x80x9d is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand gene encoding a PAKSI-related protein. Preferably, the antisense nucleic acid is capable of decreasing the activity of a PAKSI-related protein when present in a cell which normally is modulated by PAKSI. Preferably, the decrease is at least 50%, relative to a control, more preferably, 90%, and most preferably, 95-100%.
By xe2x80x9ca disease characterized by abnormal growthxe2x80x9d is meant a disease that is caused by or results in inappropriately high numbers of cells. This can be a result of inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, and lung cancer are all examples of disease characterized by abnormal cell growth.
By xe2x80x9ca disease characterized by an abnormal cytoskeleton in a specific cell typexe2x80x9d is meant a disease in which specific cells have inappropriate levels of cytoskeletal proteins or cytoskeleton associated protein, or in which such proteins are altered (e.g., by phosphorylation or dephosphorlyation). Example of diseases associated with an abnormal cytoskeleton include, for example, Alzheimer""s disease, Pick""s disease, Charcot-Marie-Tooth disease, Crohn""s disease, and dilated cardiomyopathies.